Cellular Distribution and Functions of P2 Receptor Subtypes in Different Systems

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Abstract

This review is aimed at providing readers with a comprehensive reference article about the distribution and function of P2 receptors in all the organs, tissues, and cells in the body. Each section provides an account of the early history of purinergic signaling in the organ⧸cell up to 1994, then summarizes subsequent evidence for the presence of P2X and P2Y receptor subtype mRNA and proteins as well as functional data, all fully referenced. A section is included describing the plasticity of expression of P2 receptors during development and aging as well as in various pathophysiological conditions. Finally, there is some discussion of possible future developments in the purinergic signaling field.

Introduction

In 1929 Drury and Szent-Györgyi published a seminal paper describing the potent actions of adenine compounds. Some decades later, adenosine 5′-triphosphate (ATP) was proposed as the transmitter responsible for nonadrenergic, noncholinergic (NANC) transmission in the gut and bladder and the term “purinergic” was introduced (Burnstock, 1972). The fact that ATP was recognized primarily for its important intracellular roles in many biochemical processes coupled to the intuitive feeling that such a ubiquitous and simple compound was unlikely to be utilized as an extracellular messenger fueled early resistance to this concept, even though powerful extracellular enzymes involved in the breakdown of ATP were known to be present.

Implicit in the concept of purinergic neurotransmission was the existence of postjunctional purinergic receptors; in addition, the potent actions of extracellular ATP on many different cell types also implicated membrane receptors. The first definition of purinergic receptors was put forward in 1976 (Burnstock, 1976) followed 2 years later by a proposed basis for distinguishing two types of purinoceptor, identified as P1 and P2 (for adenosine and ATP⧸adenosine diphosphate [ADP], respectively) (Burnstock, 1978). Concurrent with this, two subtypes of the P1 (adenosine) receptor were recognized (Londos 1980, Van Calker 1979); four subtypes of P1 receptors have subsequently been cloned, namely A1, A2A, A2B, and A3 (Fredholm 2001, Ralevic 1998). It was not until 1985 that the existence of two types of P2 receptors (P2X and P2Y) was proposed (Burnstock and Kennedy, 1985). The following year two further P2 purinoceptor subtypes were tentatively identified, namely a P2T receptor selective for ADP on platelets and a P2Z receptor on macrophages (Gordon, 1986). Further subtypes of P2 receptors followed, perhaps the most important being the P2U receptor that could recognize pyrimidines such as uridine triphosphate (UTP) as well as ATP (O'Connor et al., 1991). At a meeting in 1994, Williams made the point that a classification of P2 purinoceptors based on a “random walk through the alphabet” was not satisfactory, and Abbracchio and Burnstock (1994) proposed that purinoceptors should belong to two major families: a P2X family of ligand-gated ion channel receptors and a P2Y family of G protein-coupled receptors, the classification formed on the basis of transduction mechanism studies (Dubyak, 1991) and the cloning of nucleotide receptors (Brake 1994, Lustig 1993, Valera 1994, Webb 1993). This nomenclature has been widely adopted, and currently seven P2X subtypes and about eight P2Y receptor subtypes are recognized, including receptors that are sensitive to pyrimidines as well as purines (Burnstock 2003a, Ralevic 1998).

It is widely recognized that purinergic signaling is a primitive system (Burnstock, 1996a) involved in both neuronal and non-neuronal mechanisms (Abbracchio and Burnstock, 1998), including exocrine and endocrine secretion, immune responses, inflammation, pain, platelet aggregation, and endothelial-mediated vasodilatation (Burnstock 1997, Burnstock 2000a, Burnstock 2003b, Dubyak 1993, Gordon 1986, Olsson 1990). Receptors for purines and pyrimidine nucleotides are involved in both short-term signaling, such as neurotransmission and secretion, and long-term (trophic) signaling, such as cell proliferation, differentiation, and programmed cell death that occur during development and regeneration (Burnstock 2001a, Burnstock 2002, Neary 1996). P2 receptors show plasticity of expression during development and aging, following trauma or surgery, and in disease (see Section III).

This review is devoted to describing the cell and molecular biology of P2 receptor subtypes in all the body systems. Our approach has been to deal with each system in the following way: We begin with a historical introduction of the early descriptions of the actions of ATP, covering the literature up to 1994 when the first clear framework for P2 receptor subtyping into P2X ionotropic and P2Y metabotropic families was put forward (Abbracchio and Burnstock, 1994). A table follows summarizing the distribution of P2 receptor mRNA, protein, and functional receptors (receptor mRNA as seen with Northern blots, reverse transcriptase-polymerase chain reaction [RT-PCR], or in situ hybridization; protein as seen with immunostaining, Western blots, or autoradiography⧸ligand binding, and identification of functional P2 receptor subtypes as seen by pharmacology⧸electrophysiology, Ca2+ imaging, and biochemistry). The functions claimed for the receptors identified are included in the table, as well as the key references. Finally, there is a section concerned with the sources of ATP that could act on the receptors and a brief summary of the main purinergic signaling features of the system.

1. P2X receptors: Members of the existing family of ionotropic P2X1–7 receptors exhibit a subunit topology of: intracellular N- and C-termini possessing consensus binding motifs for protein kinases; two transmembrane spanning regions, the first (TM1) being involved with channel gating and the second (TM2) lining the ion pore; a large extracellular loop, with 10 conserved cysteine residues forming a series of disulfide bridges; a hydrophobic H5 region close to the pore vestibule, for possible receptor⧸channel modulation by cations (magnesium, calcium, zinc, copper, and proton ions); and an ATP-binding site, which may involve regions of the extracellular loop adjacent to TM1 and TM2 (see Fig. 1a). The P2X1–7 receptors show 30–50% sequence identity at the peptide level. The stoichiometry of P2X1–7 receptors is thought to involve three subunits that form a stretched trimer (Khakh et al., 2001).

The pharmacology of the recombinant P2X receptor subtypes expressed in oocytes or other cell types displays significant differences from the pharmacology of P2X-mediated responses in naturally occurring sites. There are several contributing factors that may explain these differences. The trimer ion pore may form heteromultimers as well as homomultimers. For example, heteromultimers of P2X2 and P2X3 receptor subtypes (P2X2⧸3) are clearly established in nodose ganglia (Lewis 1995, Radford 1997), P2X4⧸6 in central nervous system (CNS) neurons (Lê et al., 1998), P2X1⧸5 in some blood vessels (Haines 1999, Torres 1998), and P2X2⧸6 in the brain stem (King et al., 2000b). P2X7 does not form heteromultimers, and P2X6 will not form a functional homomultimer (North 2000, Torres 1999). Second, spliced variants of P2X receptor subtypes may be a contributing factor. For example, a splice variant of the P2X4 receptor, while on its own nonfunctional, can potentiate the actions of ATP through the full-length P2X4 receptors (Townsend-Nicholson et al., 1999). Third, the presence of powerful ectoenzymes that rapidly break down purines and pyrimidines in native tissues is not a factor when examining recombinant receptors (Zimmermann, 1996).

Within the P2X receptor family there are many pharmacological and operational differences between individual receptor subtypes. The kinetics of activation, inactivation, and deactivation also vary considerably among P2X receptors. Calcium permeability is high for some P2X subtypes, a property that may be functionally important. For a more specific review of P2X receptor molecular biology, cell biology, physiology, and biophysics, the reader is referred to North (2002).

2. P2Y receptors: Metabotropic P2Y1–14 receptors have a characteristic subunit topology of an extracellular N-terminus and an intracellular C-terminus, the latter possessing consensus binding motifs for protein kinases; seven transmembrane-spanning regions that help to form the ligand docking pocket; a high level of sequence homology between some transmembrane-spanning regions, in particular TM3, TM6, and TM7; the intracellular loops and C-terminus posses structural diversity among P2Y subtypes, so influencing the degree of coupling with Gq⧸11, Gs, and Gi proteins (see Fig. 1b). Each P2Y receptor binds to a single heterotrimeric G protein (typically Gq⧸11), although P2Y11 can couple to both Gq⧸11 and Gs whereas P2Y12 couples to Gi and P2Y14 to Gi⧸0. Under certain conditions P2Y receptors may form homo- and heteromultimeric assemblies, and many tissues express multiple P2Y subtypes (King et al., 2000a). P2Y receptors show a low level of sequence homology at the peptide level (19–55% identical) and, consequently, show significant differences in their pharmacological and operational profiles. P2Y1, P2Y6, and P2Y12 receptors are activated principally by nucleoside diphosphates, while P2Y2 and P2Y4 are activated mainly by nucleoside triphosphates. P2Y2, P2Y4, and P2Y6 receptors are activated by both purine and pyrimidine nucleotides and P2Y1, P2Y11, and P2Y12 receptors are activated by purine nucleotides alone. In response to nucleotide activation, recombinant P2Y receptors either activate phospholipase C (PLC) and release intracellular calcium ([Ca2+]i) or affect adenylyl cyclase and alter cAMP levels. To date there is insufficient evidence to indicate that the P2Y5, P2Y9, and P2Y10 sequences are nucleotide receptors or affect intracellular signaling cascades. Endogenous P2Y receptors show a great diversity in intracellular signaling and can activate phospholipases A2, C, and D, major excreted protein (MEP)⧸mitogen-activated protein (MAP) kinase, Rho-dependent kinase and tyrosine kinase, as well as coupling both positively and negatively to adenylyl cyclase.

At mammalian P2Y1 receptors, 2-methylthioADP (2-MeSADP) is a potent agonist (Hechler et al., 1998) and N6-methyl-2′-deoxyadenosine 3′,5′-bisphosphate (MRS 2179) a potent antagonist (Boyer et al., 1998); N6-methyl-1,5-anhydro-2-(adenin-9-yl)-2,3-dideoxy-d-arabinohexitol-4,6-bis(diammonium phosphate) (MRS 2269) and MRS 2286 have been identified as selective antagonists (Brown et al., 2000). ATP and UTP are equipotent at P2Y2 and P2Y4 receptors in the rat, but the two receptors can be distinguished with antagonists, as suramin blocks P2Y2, while Reactive Blue 2 blocks P2Y4 receptors (Bogdanov 1998b, King 1998a). P2Y6 is uridine diphosphate (UDP)-selective, while P2Y7 has been revealed to be a leukotriene receptor (Yokomizo et al., 1997). P2Y8 is a receptor cloned from frog embryos, at which all the nucleotides are equipotent (Bogdanov et al., 1997), but no mammalian homologue has been identified to date, apart from a recent report of P2Y8 mRNA in undifferentiated HL60 cells (Adrian et al., 2000). P2Y11 is unusual in that two transduction pathways can be activated, adenylate cyclase as well as inositol triphosphate (IP3), which is the second messenger system used by the majority of the P2Y receptors. The P2Y12 receptor found on platelets was not cloned until more recently (Hollopeter et al., 2001), although it has only 19% homology with the other P2Y receptor subtypes. This receptor together with P2Y13 and P2Y14 may represent a subgroup of P2Y receptors for which transduction is entirely through adenylate cyclase (Abbracchio 2003, Communi 2001a, Communi 2001b, Zhang 2002). A receptor on C6 glioma cells and possibly a receptor in the midbrain, selective for a diadenosine polyphosphate, also may operate through adenylate cyclase. An interesting question that has arisen by analogy with other G protein-coupled receptors is whether dimers can form between the P2Y subtypes. For a specific review of P2Y receptor biology and physiology, see Lazarowski (2003).

Table I summarizes the structure and properties of current receptor subtypes while Table II summarizes the current status of P2 receptor subtype agonists and antagonists.

Section snippets

Lung

ATP (probably via adenosine) has been known as a bronchodilating agent for many years (Venugopalan et al., 1986). Similarly, the presence of both vasoconstricting P2X receptors and vasodilating P2Y receptors in pulmonary vessels has long been recognized in both rats and humans (Liu 1989a, Liu 1989b).

ATP exerts various effects upon airway epithelial cells. Alveolar type II cells synthesize and secrete surfactant in response to a variety of secretagogues, of which ATP is a particularly potent

Plasticity of Purinergic Receptor Expression

There are a growing number of reports of changing expression of purinoceptors in cells and organs during development and disease (Abbracchio 1998, Burnstock 1990b, Burnstock 2001a, Hourani 1999).

Conclusions and Future Directions

Clearly functional purinoceptors are widely distributed in both neuronal and non-neuronal tissues, probably because it is a primitive (perhaps the earliest) molecular messenger. In the nervous system ATP is recognized as a cotransmitter in all peripheral and central nerve types, although its relative importance varies in different sites and with age and under pathophysiological conditions.

It seems likely that all the P2X receptor subtypes (P2X1–P2X7) have now been cloned and characterized, but

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